Project Summary/Abstract Sepsis kills more than 375,000 Americans annually. No approved drugs are available and over 30 drug candidates failed late-stage clinical trials, which highlights the urgent need for novel approaches. The long- term goal of this project is to develop a new mechanism-based, efficient and low-toxicity sepsis therapy. Triggering receptor expressed on myeloid cells 1 (TREM-1), an inflammation amplifier, is overexpressed in patients with sepsis. In mice with lipopolysaccharide (LPS)-induced endotoxic shock, blockade of TREM- 1 inhibits inflammation and protects animals from death. However, current TREM-1 inhibitors block binding of the unknown ligand to TREM-1, which highly increases the risk of their failure in clinical development. SignaBlok developed a first-in-class ligand-independent TREM-1 inhibitory peptide GF9. GF9 is well- tolerated and can be formulated into SignaBlok's lipopeptide complexes (LPC) that contain two modified peptides of human apolipoprotein (apo) A-I PE22 and PA22 and intracellularly deliver GF9 to macrophages probably due to apo A-I epitopes for binding to scavenger receptors (SR): SR-A (PE22 and PA22) and SR- BI (PA22). SR-BI is also expressed on hepatocytes and provides hepatic clearance of GF9-LPC affecting their half-life. In Phase I, we will test the hypothesis that combination and exposure of these epitopes in GF9-LPC can be optimized to provide fast and efficient GF9 delivery to macrophages by long half-life GF9- LPC. This is anticipated to provide a prompt, effective and long-lasting protection against sepsis. Phase I specific aims are to: 1) optimize GF9-LPC composition for fast and efficient delivery of GF9 to macrophages in vitro, and 2) test GF9-LPC comparatively in mouse models of polymicrobial and LPS- induced sepsis. We will generate, optimize and select the most promising GF9-LPC formulations based upon their stability, GF9 content, and rate and efficiency of murine and human macrophage uptake in vitro as well as upon their inhibitory effect on cytokine release by LPS-stimulated cells. We will determine in vivo pharmacokinetics, bioavailability, and biodistribution of these formulations, select the optimal GF9-LPC formulation and test two doses of it in mice with sepsis induced by cecal ligation and puncture (CLP) or LPS. The use of two animal models will address the known differences in their patterns of cytokines and pharmacomodulation. We will determine both systemic inflammation (by measuring serum and tissue cytokines) and animal survival. Comprehensive histology will be performed to analyze organ protection and/or potential non-specific toxicity for organs/tissues. GF9 biodistribution will be analyzed using LC-MS. It is anticipated that the Phase I study will identify novel, first-in-class, low toxic TREM-1 inhibitors that will provide a powerful platform for development of effective and safe sepsis therapies, thereby improving sepsis treatment and increasing patient survival. If successful, Phase I will be followed in Phase II by toxicology, ADME, pharmacology and CMC studies, filing an IND and subsequent evaluation in humans.